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We examined the influence of drought stress during grain filling on grain yield to investigate changes in assimilates in sink and source organs. When plants were subjected to drought stress from the start of grain filling until harvest, the photosynthetic rate rapidly decreased. Grain dry mass during maturation was not significantly different between the control and drought-stressed plants. Under drought stress conditions, starch content in source organs (peduncle, leaf, petiole, stem, and root) was significantly lower than in corresponding organs of control plants; the greatest difference was seen in leaves. Consistent with this observation, α- and β-amylase activities in leaves significantly increased within the first 6 d of drought stress. We conclude that in cowpea subjected to drought stress during grain filling, the grain yield is maintained, despite a dramatic decrease in photosynthetic rate, by translocation of photoassimilates from source organs.

Environmental stresses can reduce crop yield and quality considerably. Plants protect cell metabolism in response to abiotic stresses at all stages of their life cycle, including seed production. As the production of vigorous seeds is important to both yield and crop growth, we analyzed causes of yield loss and reduced grain quality in staple crops exposed to environmental stresses such as drought and temperature extremes, with a focus on the remobilization of nutrients and water status during seed filling. Because water is one of the factors that limit seed development, seeds must have mechanisms that allow them to withstand water loss during seed maturation. In addition, analysis of the effects of reactive oxygen species (ROS) on transcription regulation and signaling should help to elucidate the regulation of seed dormancy and germination. In this review, we focus on nutrient remobilization, water mobility, plant hormones (gibberellins, abscisic acid, and ethylene), and ROS in sink and source organs and describe how rice, wheat, barley, soybean, and cowpea plants control seed maturation and germination under environmental stresses.

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Environmental stresses can reduce crop yield and quality considerably. Plants protect cell metabolism in response to abiotic stresses at all stages of their life cycle, including seed production. As the production of vigorous seeds is important to both yield and crop growth, we analyzed causes of yield loss and reduced grain quality in staple crops exposed to environmental stresses such as drought and temperature extremes, with a focus on the remobilization of nutrients and water status during seed filling. Because water is one of the factors that limit seed development, seeds must have mechanisms that allow them to withstand water loss during seed maturation. In addition, analysis of the effects of reactive oxygen species (ROS) on transcription regulation and signaling should help to elucidate the regulation of seed dormancy and germination. In this review, we focus on nutrient remobilization, water mobility, plant hormones (gibberellins, abscisic acid, and ethylene), and ROS in sink and source organs and describe how rice, wheat, barley, soybean, and cowpea plants control seed maturation and germination under environmental stresses.

Seed dormancy is one of the adaptive responses in the plant life cycle and an important agronomic trait. Reactive oxygen species (ROS) release seed dormancy and promote seed germination in several cereal crops; however, the key regulatory mechanism of ROS-mediated seed dormancy and germination remains controversial. Here, we focused on the relationship between hydrogen peroxide (a ROS) and abscisic acid (ABA) in dormant and non-dormant barley seeds. The hydrogen peroxide (H2O2) level produced in barley seed embryos after imbibition was higher in non-dormant seeds than in dormant seeds. H2O2 regulated the ABA content in the embryos through ABA-8′-hydroxylase, an ABA catabolic enzyme. Moreover, compared with non-dormant seeds, in dormant seeds the activity of NADPH oxidase, which produces ROS, was lower, whereas the activity of catalase, which is a H2O2 scavenging enzyme, was higher, as was the expression of HvCAT2. Furthermore, precocious germination of isolated immature embryos was suppressed by the transient introduction of HvCAT2 driven by the maize (Zea mays) ubiquitin promoter. HvCAT2 expression was regulated through an ABA-responsive transcription factor (HvABI5) induced by ABA. These results suggest that the changing of balance between ABA and ROS is active in barley seed embryos after imbibition and regulates barley seed dormancy and germination.

In order to establish methodology for population estimation of Asiatic black bears using the hair-trapping method, hair-trapping devices were located within a study area, and basic data were collected to determine the optimal survey period for hair sampling. Bear hairs collected were classified as three types (guard hair; G, underfur; U, and intermediate hair; I) depending on hair-bulb thickness. After DNA extraction, six microsatellite loci and amelogenin locus were amplified in order to identify individual bears and sex, respectively. The number of hairs collected by hair-trapping devices decreased dramatically in late August, while those that had lost part of the hair bulb increased after September. The success rate of genetic analysis was over 90% prior to the month of July and rapidly decreased in August. Based on the results of the generalized linear model, it was determined that sampling session had the most significant impact on the success rate of genetic analysis of all the explanatory...

Aims: Inducer of CBF Expression 1 (ICE1), which is one of basic Helix-Loop-Helix type transcription factors, has important roles in regulation of cold stress-indiced genes of plants. Sample: To investigate functions of tomato ICE1 in cold and salt tolerance, c index (SI), immunochemical assay of endogenous ICE1 protein and RT-PCR of coldinducible genes were conducted with tomato plants. Methodology: Tomato plants grown for 4 weeks were subjected to cold (4oC) and salt (0.2 M NaCl) in the presence or the absence of cell signaling inhibitors. An antibody was raised against ICE1 specific epitoipe. Immunoblot with the anti-ICE1 antibody was carried out with extractions of tomato plants treated by cold and salt stresses. The expression profiles of tomato ICE1 (SlICE1) and other cold-inducible genes including LeCBF1/2/3 and SlTPS1 were analyzed by semi-quantitative RT-PCR. Results: An ICE1-related protein with molecular masses of approximately 55 is induced in tomato plant under chilling and salt stresses. The expression of a tomato ICE1 gene (SlICE1) under chilling stress was maintained at a constant level in contrast to the protein Original Research Article American Journal of Experimental Agriculture, 4(7): 785-796, 2014 786 level. Chilling stress sequentially upregulated tomato CBF homolog, SlCBF1 and trehalose-6-phosphate synthase (SlTPS1). Based on the whole genome database of tomato, cis-elements potentially binding to ICE1 and CBF were located in upstream sequences in promoter regions of SlCBF1 and SlTPS1, respectively. Conclusion: Tomato ICE1 homolog mediates the expression of SlCBF1in a cold-stressinduced transcription factor cascade via binding to ICE1-specific cis-elements, leading to induction of cold tolerance by trehalose synthesis.

Low irradiance (shading) during the early phase of grain filling in rice ( Oryza sativa L.) results in low grain yield and quality. The effects of shading on partitioning of assimilated carbon within the panicle and the whole plant have been reported, but its effect on the ability to transport assimilated carbon has not been examined in detail. The objective of this study was to investigate the effects of shading on the transport of assimilated carbon and on the expression of the sucrose transporter gene OsSUT1 during the early phase of grain filling. Shading of the whole plant for 14 days after flowering reduced grain dry weight and OsSUT1 expression in grains, flag leaf, flag leaf sheath, first leaf blade, first leaf sheath, second leaf blade, and stem (but not in second leaf sheath), although initially (after 1 week) the expression in grains and second leaf sheath increased. We also shaded sink (panicle), source (leaf and leaf sheath), or whole plant from 7 to 14 days after flowering and examined the expression of OsSUT1 in grains and panicle branches. In grains, OsSUT1 expression increased in all shading treatments. In branches, OsSUT1 expression decreased upon shading of the whole plant or sink, but increased upon source shading. Additionally, 1000-grains dry weight and ratio of perfect grain significantly decreased upon shading of the whole plant, but not sink or source. These results suggest that the decrease in grain yield and quality by shading during the early phase of grain filling is caused not only by shortage of leaf-derived photoassimilates, but also by suppression of OsSUT1 in sink and source.

High temperature reduces the grain quality of rice, a situation likely to become more frequent because of global warming. We studied the effects of high-temperature stress on grain quality of heat-tolerant cultivar ‘Genkitsukushi’ and heat-sensitive cultivar ‘Tsukushiroman’. When day/night temperatures were 31/26 °C from heading until maturity, the grain quality of ‘Genkitsukushi’ was rated at the first inspection grade (high quality), whereas ‘Tsukushiroman’ showed a remarkable increase in the percentage of white immature kernels (low quality). Nonstructural carbohydrate content in the stem of ‘Genkitsukushi’ the early maturation was significantly higher than in ‘Tsukushiroman’ and greatly decreased under high temperature. From 14 to 21 days after heading, the expression of the sucrose transporter gene, OsSUT1 , was higher in the stem of ‘Genkitsukushi’ grown under high temperature than in ‘Tsukushiroman’. In addition, the expression of OsSUT1 in the grains of ‘Genkitsukushi’ was significantly higher than in ‘Tsukushiroman’ during the ripening period. These results indicate that sugar transport functions more effectively in ‘Genkitsukushi’ than in ‘Tsukushiroman’, and that the effectiveness of sugar transport contributes to maintaining high grain quality in ‘Genkitsukushi’ under high-temperature conditions.

Heat stress induces expression of a set of thermotolerance-related genes in plants. We focused on rice (Oryza sativa L.) homologs of the gene family that encodes galactinol synthase (OsGolS), which is closely related to the Arabidopsis thaliana galactinol synthase (AtGolS) family whose expression is induced under various stresses. OsGolS1 was highly up-regulated compare to the level of OsGolS2 in re- sponse to heat stress. Interestingly, OsGolS1 was also up-regulated by treatment with the Hsp90 inhibitor, geldanamycin (GDA). Expression profiles of OsGolS1 were correlated to those of OsHsfA2 under the GDA treatments. Treatment with GDA increased expression of OsHsfA2, but marginally increased or did not change OsHsfA1 expression. Notably, gel shift assay indicated that OsHsfA2 binds directly to OsGolS1 promoter region and that OsHsfA1 also binds to the promoter regions of OsHsfA2. Both OsHsfA2 and OsGolS1 were dramatically induced in response to heat stress. Accordingly, galactinol and raffinose contents in rice seedlings increased significantly following the induction of OsGolS1. Pre-treatment of rice seedlings with raffinose or GDA improved their thermotolerance. These results suggest that OsGolS1 plays an important role in response to heat stress, possibly via the transcription cascade of OsHsfA1-OsHsfA2 that leads to galactinol and raffinose accumulation, and that the increased content of these carbohydrates is a key response factor for rice seedlings to enhance thermotolerance.

In higher plants, autophagy is bulk degradation process in vacuole necessary for survival under nutrient-limited conditions and plays important roles in senescence, development and pathogenic response, etc. Cowpea is one of the most important legume crops in semi-aride region, which is highly tolerant to drought stress. Changes of photoassimilate status by drought stress and/or sink-source balance appeared to affect autophagy and senescence of leaf in cowpea. Accordingly, we focused on roles of sucrose signal in autophagy and amino acid recycling in cowpea. Effects of starvation stress on the expression of autophagy-related genes (ATGs) and amino acid catabolism-related genes in cowpea [Vigna unguiculata (L.) Walp] were examined by Reverse transcription-polymerase chain reaction (RT-PCR) and anti-ATG8i specific antibody. Sucrose starvation stress enhanced the expression levels of VuATG8i, VuATG8c and VuATG4 incowpea seedlings. The expressions of amino acid catabolism related genes, such as asparagine synthase (VuASN1), proline dehydrogenase1 (VuProDH) and branched chain amino acid transaminase (VuBCAT2), are also up-regulated under the sucrose starvation. In contrast, high sucrose condition suppressed autophagy and the expressions of ATGs. These results indicate that sucrose starvation stress stimulates both autophagy and amino acid catabolism by regulation of ATGs and VuBCAT2. It is conceivable that sucrose starvation stress enhances autophagy in cowpea, possibly via branched chain amino acid level regulated by the starvation-induced BCAT.

High temperatures during rice grain ripening reduced yield and grain quality. The proportion of milky white grains was 43.6 % at 30 °C but only 6.5 % at 25 °C. Grain filling was initially faster at 30 °C and finished earlier, and the final dry matter content was less, than at 25 °C. High temperature strongly suppressed the expression of the sucrose transporter gene OsSUT1 and starch synthesis-related genes SuSy2, AGPS2b, BEIIb and Granule-bound starch synthase in grains during early grain filling; the transcription levels of OsSUT1 at 14 days after flowering (DAF) were about 60 % lower in grains, flag leaf blade, flag leaf sheath and first leaf sheath. These facts are possibly involved in the earlier termination of grain filling at 21 DAF, following the rapid rise of grain dry weight from 0 to 7 DAF, due to possible reduction in assimilate supply via OsSUT1 under the high temperature. When panicles were partly clipped, the resultant increase in assimilate supply to the remaining grains significantly upregulated the expression of OsSUT1 and the starch synthesis-related genes at 14 DAF, which consequently accelerated starch accumulation in the grains and ultimately increased the grain weight of remaining grains at 30 °C. These results indicate that high temperature during grain filling reduces grain yield and quality by changing the expression of OsSUT1 and starch synthase-related genes, resulting in earlier ripening due to hastened or premature assimilate supply to grains.

Non-invasive DNA genotyping using hair samples has become a common method in population surveys of Asiatic black bears (Ursus thibetanus) in Japan; however, the accuracy of the genotyping data has rarely been discussed in empirical studies. Therefore, we conducted a large-scale pilot study to examine genotyping accuracy and sought an efficient way of error-checking hair-trapping data. We collected 2,067 hair samples, successfully determined the genotypes of 1,245 samples, and identified 295 individuals. The genotyping data were further divided into 3 subsets of data according to the number of hairs used for DNA extraction in each sample (1–4, 5–9, and ≥10 hairs), and the error rates of allelic dropout and false alleles were estimated for each subset using a maximum likelihood method. The genotyping error rates in the samples with ≥10 hairs were found to be lower than those in the samples with 1–4 and 5–9 hairs. The presence of erroneous genotypes among the identified individuals was further checked using a post hoc goodness-of-fit test that determined the match between the expected and observed frequencies of individual homozygotes at 0–6 loci. The results indicated the presence of erroneous genotypes, possibly as a result of allelic dropout, in the samples. Therefore, for improved accuracy, it is recommended that samples containing ≥10 hairs should be used for genotyping and a post hoc goodness-of-fit test should be performed to exclude erroneous genotypes before proceeding with downstream analysis such as capture-mark-recapture estimation.

Reactive oxygen species (ROS) act as signal molecules for a variety of processes in plants. However, many questions about the roles of ROS in plants remain to be clarified. Here, we report the role of ROS in gibberellin (GA) and abscisic acid (ABA) signaling in barley (Hordeum vulgare) aleurone cells. The production of hydrogen peroxide (H2O2), a type of ROS, was induced by GA in aleurone cells but suppressed by ABA. Furthermore, exogenous H2O2 appeared to promote the induction of α-amylases by GA. In contrast, antioxidants suppressed the induction of α-amylases. Therefore, H2O2 seems to function in GA and ABA signaling, and in regulation of α-amylase production, in aleurone cells. To identify the target of H2O2 in GA and ABA signaling, we analyzed the interrelationships between H2O2 and DELLA proteins Slender1 (SLN1), GA-regulated Myb transcription factor (GAmyb), and ABA-responsive protein kinase (PKABA) and their roles in GA and ABA signaling in aleurone cells. In the presence of GA, exogenous H2O2 had little effect on the degradation of SLN1, the primary transcriptional repressor mediating GA signaling, but it promoted the production of the mRNA encoding GAMyb, which acts downstream of SLN1 and involves induction of α-amylase mRNA. Additionally, H2O2 suppressed the production of PKABA mRNA, which is induced by ABA:PKABA represses the production of GAMyb mRNA. From these observations, we concluded that H2O2 released the repression of GAMyb mRNA by PKABA and consequently promoted the production of α-amylase mRNA, thus suggesting that the H2O2 generated by GA in aleurone cells is a signal molecule that antagonizes ABA signaling.

Many abiotic and biotic stresses can reduce plant growth and development. Low temperature is one of the most harmful abiotic stresses, particularly for plants that are tropical or subtropical in origin. To elucidate the molecular mechanisms underlying the cold-stress response, components involved in the signal transduction of cold stress have been c haracterized. In this study, we characterized a basic helix-loop-helix (bHLH) transcription factor encoding gene, SlICE1, from tomato (Solanum lycopersicum), which shows similarity with Arabidopsis ICE1. �e expression of SlICE1 was observed in younger leaves, �owers, and green and red fruits. To characterize the function of SlICE1, overexpressing toma to lines were produced. SlICE1-overexpressing tomatoes exhibited chilling tolerance, and SlICE1 enhanced the expression of cold- responsive genes, such as SlCBF1 and SlDRCi7, as well as accumulation of ascorbic acid. �e SlICE1 protein was degraded aer cold treatment. �ese results indicate that SlICE1 enhances cold tolerance in tomatoes.

Floral and reproductive organs of higher plants are relatively sensitive to biotic and abiotic stresses compared with the vegetative organs. Calcineurin B-like molecule (CBL) interacting protein kinase (CIPK) has appeared to be involved in acquired tolerance and acclimation under environmental stresses such as salinity, drought and chilling. Semi-quantitative RT-PCR using the vegetative and reproductive organs of tomato Micro-Tom (Solanum lycopersicum L.) at the various developmental stages indicated that SlCIPK2 was expressed specifically in the floral organ. An anti-CIPK specific antibody recognized the recombinant SlCIPK2 specifically and cross-reacted to a CIPK-related polypeptide at a significant level in flower, particularly in stamen. The flower specific CIPK was tightly associated with the microsomes. In vitro pull-down assay of the recombinant SlCIPK2 showed that SlCIPK2 interacts with SlCBLs and stress-responsive transcription factors, SlERF7, SlCBF1 and SlAREB1. The present data suggested that the flower-specific CIPK, SlCIPK2, was involved in calcium signaling in tomato via CBLs and stress tolerance possibly mediated by the stress-responsive transcription factors in stamen.

ADP–glucose pyrophosphorylase (AGPase) catalyzes the first limiting step in starch biosynthesis in plants. However, the direct transcriptional activator of the AGPase genes has not yet been determined. We have isolated a WRKY transcription factor cDNA, AtWRKY20, from Arabidopsis thaliana and purified the corresponding protein. Transient expression of AtWRKY20 by particle bombardment enhanced expression of the promoter of ApL3, encoding a sugar-inducible AGPase large subunit gene of A. thaliana, in leaves of A. thaliana. AtWRKY20 bound to the ApL3 promoter in vitro. The expression of AtWRKY20 was strongly induced by sucrose or, to a lesser extent, by mannitol, and the expression pattern of the ApL3 gene mimicked that of the AtWRKY20 gene. Transient expression experiments demonstrated that AtWRKY20 also activated the promoter of Koganesengan ibAGP1 encoding an AGPase small subunit gene of sweet potato var. Koganesengan. A 5’–end deletion analysis revealed a negative regulatory region from –1371 to –641 and a positive regulatory region from –640 to –180 in the Koganesengan ibAGP1 promoter. AtWRKY20 interacted directly with the region between positions –623 and –490 in the Koganesengan ibAGP1 promoter. These results suggest that AtWRKY20 functions directly as a transcriptional activator of the ApL3 promoter and regulates the expression of ApL3 induced by sucrose or osmoticum in A. thaliana. Moreover, AtWRKY20 can enhance the expression of the Koganesengan ibAGP1 promoter directly in sweet potato.

Some Asian black bears inhabiting Japan are known to invade residential areas, a phenomenon known as mass intrusion. Although mass intrusion occurs in autumn, it can result in genetic mingling. We examined the influence of intrusion on the genetic structure of black bears. Tissue samples were collected from invasive bears in central Japan in the autumn of two mass-intrusion years, 2004 and 2006. We also set hair traps overlapping an area where tissue samples were collected in the summers of 2005, 2006, and 2008. The genotypes of eight microsatellite loci were determined by PCR. Pairwise relatedness (rxy ) was negatively correlated with distance among resident bears, whereas no such relationship was observed among invasive bears. Spatial autocorrelation analysis of the normal bears in 2006 revealed a significantly positive rc value within the 0–65-km-distance class, whereas that of invasive bears in 2006 revealed significantly positive rc values between the 0–15- and 0–65-km-distance classes. We ...

Autophagy appears to function in bulk protein degradation and N remobilization in senescence. Depodding of soybean suppresses N remobilization from leaves, leading to “green stem syndrome”. Expression of autophagy-related genes (ATGs) and N contents of the leaves and stem was analyzed in soybean plants from none (control), half (50% depodding) and all (100% depodding) of flowers and pods were removed at the reproductive 5 stage. Total N content and SPAD in leaves of the plant after 100% depodding were retained at a constant level and those after 50% depodding gradually decreased from 4 to 5 wk after depodding, while those in the control plants rapidly decreased during this period. Expression of GmATG8c and GmATG8i in leaves peaked at 4 wk in control while the gene expression increased gradually after 50% and 100% depodding. The transient up-regulation of ATGs and the decline of leaf N content and SPAD occurred simultaneously. These results indicated that ATGs are involved in leaf senescence and N remobilization from leaf to pod.

Cold stress on crops results in severe yield losses through growth retardation and irreversible damage. Recently, Inducer of CBF Expression 1 (ICE1) was identified as the master regulator inducing dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF)-type transcriptional factors involved in the signaling of cold and osmotic stress in Arabidopsis. To examine whether rice ICE homologs function in cold acclimation via regulation of rice DREB homologs in response to cold stress, we assessed a polypeptide epitope containing an ICE-specific motif. Chilling stress on rice seedlings induced two ICE-related proteins with molecular masses of approximately 55 and 40 kDa. These sizes are consistent with those predicted for OsICE1 and OsICE2, respectively. In contrast to the proteins, cold stress had little or no effect on the expression of OsICE1 and OsICE2. Semi-quantitative RT-PCR indicated that both were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6- phosphate phosphatase (OsTPP1). Trehalose treatment enhanced the cold tolerance of seedlings. These results suggest that OsICE homologs function in transcriptional regulation at upstream of a cold-stress-induced transcription factor cascade involving OsDREB1B and OsHsfA3, leading to cold acclimation, possibly involving trehalose synthesis.

To examine whether silica bodies are essential for silicon-enhanced growth of rice seedlings, we investigated the response of rice, Oryza sativa L., to silicon treatment. Silicic acid treatment markedly enhanced the SPAD (soil plant analytical development) values of leaf blades and the growth and development of leaves and lateral roots in cvs. Hinohikari and Oochikara, and a low-silicon mutant, lsi1. Combination of ethanol–benzene displacement and staining with crystal violet lactone enabled more detailed histochemical analysis to visualize silica bodies in the epidermis under bright-field microscopy. Supply of silicon induced the development of motor cells and silica bodies in epidermal cells in Hinohikari and Oochikara but not or marginal in lsi1. X-ray analytical microscopy detected silicon specifically in the leaf sheath, the outermost part of the stem, and the leaf blade midrib, suggesting that silicon is distributed to tissues involved in maintaining rigidity of the plant to prevent lodging, rather than being passively deposited in growing tissues. Silicon supplied at high dose accumulated in all rice seedlings and enhanced growth and SPAD values with or without silica body formation. Silicon accumulated in the cell wall may play an important physiological role different from that played by the silica deposited in the motor cell and silica bodies.

Autophagy functions in bulk degradation of proteins and organelles for nutrient recycling. Recent evidence suggests that a set of autophagy-related (ATG) genes are induced under starvation, senescence and various abiotic stresses. To understand how these genes function in plants, expression profiles of soybean ATG homologs, GmATG8s, GmATG4, GmATG9, GmATG12 and GmATG18a, were examined using seedlings of Glycine max (L.) Merr. cv. Fukuyutaka) subjected to various nutrient conditions. Among them, GmATG8i mRNA was induced at higher level than any other ATGs under starvation. Immuoblot with a specific antibody raised against GmAtg8i indicated that endogenous GmAtg8i associates to microsomes of soybean seedling extracts. In the presence of vacuolar protease inhibitors, significant accumulation of anti- GmAtg8i was observed and mRNA level of GmATG8i increased in higher level than in the absence of the inhibitors. These results suggest that in soybean gene expression of ATG8i is regulated by both environmental nutrient conditions and intracellular nutrient recycling via proteolysis in vacuole.

Rice productivity is related to the ability of plants to adapt to heat stress. The heat-tolerant cultivars ‘Nikomaru’ and ‘Chikushi 64’ and heat-sensitive cultivar ‘Hinohikari’ were grown at 30ºC and 25ºC for 49 days after flowering. At 30ºC, only a few white immature kernels were produced in ‘Nikomaru’ and ‘Chikushi 64’, but about 22% of grains had immature kernels in ‘Hinohikari’. The high temperature(30ºC) caused no significant changes in grain dry weight, water content, and the NMR T1 value during the early ripening stage in ‘Nikomaru’ and ‘Chikushi 64’. It also did not affect grain development, especially with respect to the nucellar epidermis, in ‘Nikomaru’ and ‘Chikushi 64’, but caused clear cessation of development of the nucellar epidermis at 14 days after flowering in ‘Hinohikari’. In addition, high temperature decreased the amylose content and increased hardnessvs. adhesion ratio of cooked rice in both ‘Nikomaru’ and ‘Chikushi 64’ resulting a softer, less sticky texture, but not in ‘Hinohikari’. The maximum viscosity and breakdown values were increased, and final viscosity decreased at 30ºC in all three cultivars. These results suggested that starch in the endosperm of grains changed from a fluid state to a doughy state more slowly in ‘Nikomaru’ and ‘Chikushi 64’ than in ‘Hinohikari’, in which the water content and NMR relaxation time decreased, and transported assimilates accumulated slowly during grain development.

Hydrogen peroxide (H(2)O(2)) promotes seed germination of cereal plants and ascorbic acid which acts as antioxidant suppresses the germination of wheat seeds, but the role of H(2)O(2) scavenging on germination during seed maturation has not been demonstrated. We investigated relationship of germination, ascorbate, H(2)O(2) scavenging enzymes and sensitivity to ascorbic acid (AsA) maturing seeds of two typical wheat (Triticum aestivum L.) cultivars, cvs. Shirogane-Komugi and Norin61. Shirogane-Komugi had marked high germination ability than Norin61 during seed maturation. Although the H(2)O(2) content had no difference in the two culti-vars, sensitivity to AsA of Norin61 seeds was higher than that of Shirogane-Komugi seeds during seed maturation. The sensitivity to AsA closely correlated with germination characteristic in the two cultivars. Especially, at 28 days after pollination (DAP), sensitivity to AsA in Norin61 seeds was remarkably high. At that stage, no significant differences were observed in endogenous AsA level, ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) activities in the two cultivars. However, catalase (CAT, EC 1.11.1.6) activity and CAT mRNA in Norin61 were remarkably higher than in Shirogane-Komugi. Sensitivity to AsA at 35 and 42 DAPs kept high levels in Norin61, and endogenous AsA and CAT activity in the seeds were significantly higher than in Shirogane-Komugi. These results revealed a direct correlation between germination and antioxidant sensitivity during the developmental stages of wheat seeds.

We isolated a calcineurin B-like protein (CBL)-interacting protein kinase (EC 2.7.11.1) homolog (VuCIPK1) from cowpea (Vigna unguiculata (L.) Walp.) with significant similarity to AtCIPK3 (86% amino acid identity), which is involved in Ktransport. VuCIPK1 mRNA was detected in the whole plant by RT-PCR. Any significant change in VuCIPK1 mRNA levels was not detected when cowpea was subjected to various environmental stresses. Immunoblot assay with an anti-VuCIPK1 specific antibody and an anti-CBL antibody showed that immunologically CIPK- and CBL-related polypeptides were preferentially associated with membrane fractions of cowpea. The anti-VuCIPK1 antibody cross-reacted to two different CIPK-related polypeptides with relative molecular masses of 51 kDa in root and 55 kDa in mature leaf. Immunoprecipitation assay by anti-VuCIPK1 antibody using cowpea leaf extracts showed that endogenous VuCIPK1 was autophosphorylated on the threonine residues in response to salt stress. The recombinant VuCIPK1 phosphorylated casein in the presence of Mn 2� . These observations suggest that membrane-associated VuCIPK1 is involved osmotic stress- activated kinase regulated by its phosphorylation status.

Reactive oxygen species (ROS) promote the germination of several seeds, and antioxidants suppress it. However, questions remain regarding the role and production mechanism of ROS in seed germination. Here, we focused on NADPH oxidases, which produce ROS. After imbibition, NADPH oxidase mRNAs were expressed in the embryo and in aleurone cells of barley seed; these expression sites were consistent with the sites of ROS production in the seed after imbibition. To clarify the role of NADPH oxidases in barley seed germination, we examined gibberellic acid (GA) / abscisic acid (ABA) metabolism and signaling in barley seeds treated with diphenylene iodonium chloride (DPI), an NADPH oxidase inhibitor. DPI significantly suppressed germination, and suppressed GA biosynthesis and ABA catabolism in embryos. GA, but not ABA, induced NADPH oxidase activity in aleurone cells. Additionally, DPI suppressed the early induction of α-amylase by GA in aleurone cells. These results suggest that ROS produced by NADPH oxidases promote GA biosynthesis in embryos, that GA induces and activates NADPH oxidases in aleurone cells, and that ROS produced by NADPH oxidases induce α-amylase in aleurone cells. We conclude that the ROS generated by NADPH oxidases regulate barley seed germination through GA / ABA metabolism and signaling in embryo and aleurone cells.

An in-gel kinase assay revealed that homogenates of Closterium ehrenbergii contain Ca2+-dependent protein kinases (CDPKs) ranging in relative molecular mass from 47 to 60 kDa, that phosphorylated histone H1, myelin basic protein, and casein as substrates in a Ca2+-dependent manner. Immunoblotting and immunoprecipitation assays of Closterium extracts using anti-Dunaliella CDPK serum showed that the antibody cross-reacted with a Closterium CDPK with a relative molecular mass of 55 kDa in the soluble fraction. These results indicate that Closterium possesses CDPK-related polypeptides: one is a 55-kDa CDPK that is immunologically related to Dunaliella CDPK and the others have various molecular masses.

Salicylic acid (SA)-induced protein kinase (SIPK) in Nicotiana tabacum L. is activated in response to various stimuli. We assessed the response of SIPK in N. benthamiana. leaves to ultraviolet-C (UV-C) and SA stress. Infiltration of Agrobacterium tumefaciens EHA105 cells containing pBE2113-GST-SIPK into leaves gave transient production of glutathione-S transferase (GST)-fused SIPK. Irradiation of agroinfiltrated leaves with UV-C (254 nm) activated GST-SIPK and endogenous protein kinases with a molecular mass of about 45 kDa, close to that of SIPK simultaneously. These results suggest that SIPK is involved in stress signalling in Nicotiana plants under UV-C irradiation.

On the basis of the genome sequence, the unicellular motile cyanobacterium Synechocystis sp. PCC 6803 harbors seven putative genes for eukaryotic-type protein kinase belonging to Pkn2 subfamily ( spkA approximately spkG). Previously, SpkA was shown to have protein kinase activity and to be required for cell motility. Here, the role of the spkB was examined. The spkB gene was expressed in Escherichia coli as a fusion protein with His-tag, and the protein was purified by Ni(2+) affinity chromatography. The eukaryotic-type protein kinase activity of the expressed SpkB was demonstrated as autophosphorylation to itself and phosphorylation of the general substrate proteins. SpkB showed autophosphorylation activity in the presence of both Mg(2+) and Mn(2+), but not in Ca(2+). Phenotype analysis of spkB disruptant of Synechocystis revealed that spkB is required for cell motility, but not for phototaxis. These results suggest that SpkB is the eukaryotic-type protein kinase, which regulates cellular motility via protein phosphorylation like SpkA.

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.

Activation of a 40 kDa protein kinase in Dunaliella tertiolecta was detected by in-gel kinase assay when the cells were subjected to various forms of stress, such as heat shock, acidic stress and H2O2. The substrate specificity of the 40 kDa kinase resembles that of HAP kinase (high osmotic pressure-activated protein kinase). An anti-phosphotyrosine monoclonal antibody did not co-precipitate with the 40 kDa kinase in the cell extracts of Dunaliella subjected to stress. Treatment of Dunaliella cells with mastoparan or AlF4-, trimeric G-protein activators, stimulated another protein kinase with a relative molecular mass of 46 kDa. This protein kinase has a different substrate specificity from that of HAP kinase and the stress-activated 40 kDa kinase. These results suggest that the stress-activated 40 kDa kinase is closely related to HAP kinase and that the 46 kDa kinase is associated with a putative trimeric G-protein.

A cDNA clone encoding a Ca2 +-dependent protein kinase (DtCPK1) with a calculated molecular mass of 65,746 Da was isolated by sequential immuno- and hybridization-screening from a cDNA library of the halotolerant green alga, Dunaliella tertiolecta Butcher (Chlorophyceae). Primary structure analysis of DtCPK1 revealed a long variable domain preceding a catalytic domain, an autoinhibitory junction domain, and a C-terminal calmodulin-like domain containing 4 EF-hand motifs. Database searches showed that DtCPK1 has a high similarity to CCK1, a CDPK from the green alga, Chlamydomonas eugamentos Moewus. The N-terminal long variable domain of DtCPK1 contains neither the N-myristoylation motif, which is found in many CDPKs, nor the PEST motif, which is associated with rapid protein turnover and found in one CDPK subfamily. However, a putative Ca2 +-dependent lipid binding domain that might be responsible for the association of cytosolic DtCPK1 with the cell membrane was identified in the variable domain. Three CDPKs, with molecular masses of 62, 54, and 47 kDa respectively, were observed in an in-gel protein kinase assay of D. tertiolecta cells extract. No change in the activities of these CDPKs were observed for up to 30 min after D. tertiolecta cells had been subjected to a hypoosmotic shock. An antibody raised against a CDPK purified from D. tertiolecta and used to isolate the DtCPK1 cDNA clone cross-reacted strongly with the 62-kDa CDPK but weakly with the 54-kDa CDPK in a Western blot, indicating that the 62-kDa CDPK is identical to DtCPK1. There was no change in the intensity of these bands after hypoosmotic shock, implying that the cellular level of the enzyme protein is not associated with hypoosmotic shock. These results indicate that CDPK is activated only by the increase in cytosolic-free Ca2 + concentration in vivo.

The homeostasis of gibberellins (GAs) is maintained by negative-feedback regulation in plant cells. REPRESSION OF SHOOT GROWTH (RSG) is a transcriptional activator with a basic Leu zipper domain suggested to contribute GA feedback regulation by the transcriptional regulation of genes encoding GA biosynthetic enzymes. The 14-3-3 signaling proteins negatively regulate RSG by sequestering it in the cytoplasm in response to GAs. The phosphorylation on Ser-114 of RSG is essential for 14-3-3 binding of RSG; however, the kinase that catalyzes the reaction is unknown. Recently a Ca(2+)-dependent protein kinase (CDPK) was identified as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of the Ser-114 of RSG. Our results suggest that CDPK decodes the Ca(2+) signal produced by GAs and regulates the intracellular localization of RSG in plant cells.

Tumor necrosis factor (TNF) elicits a diverse array of inflammatory responses through engagement of its type-1 receptor (TNFR1). Many of these responses require de novo gene expression mediated by the activator protein-1 (AP-1) transcription factor. We investigated the mechanism by which TNFR1 recruits the stress-activated protein kinases (SAPKs) and the p38s, two mitogen-activated protein kinase (MAPK) families that together regulate AP-1. We show that the human SPS1 homologue germinal center kinase (GCK) can interact in vivo with the TNFR1 signal transducer TNFR-associated factor-2 (TRAF2) and with MAPK/ERK kinase kinase 1 (MEKK1), a MAPK kinase kinase (MAPKKK) upstream of the SAPKs, thereby coupling TRAF2 to the SAPKs. Receptor interacting protein (RIP) is a second TNFR signal transducer which can bind TRAF2. We show that RIP activates both p38 and SAPK; and that TRAF2 activation of p38 requires RIP. We also demonstrate that the RIP noncatalytic intermediate domain associates in vivo with an endogenous MAPKKK that can activate the p38 pathway in vitro. Thus, TRAF2 initiates SAPK and p38 activation by binding two proximal protein kinases: GCK and RIP. GCK and RIP, in turn, signal by binding MAPKKKs upstream of the SAPKs and p38s.

To address roles of bZIP transcription factors on regulation of amino acid catabolism under autophagy-induced plant cells, we examined the effect of nutrient starvation on the expression of low energy stress-related transcription factor homologs, GmbZIP53A and GmbZIP53B, and amino acid catabolism-related genes in soybean (Glycine max (L.) Merr.). Sucrose starvation treatment significantly enhanced the expressions of GmbZIP53A, but not GmbZIP53B asparagine synthase (GmASN1), proline dehydrogenase1 (GmProDH), and branched chain amino acid transaminase 3 (GmBCAT3). GmbZIP53-related immunoreactive signals were upregulated under severe starvation with sucrose starvation and protease inhibitors, while 3% sucrose and sucrose starvation had no or marginal effects on the signal. Profiles of induction of GmASN1, GmProDH and GmBCAT3 under various nutrient conditions were consistent with the profiles of GmbZIP53 protein levels but not with those of GmbZIP mRNA levels. These results indicate that GmbZIP53 proteins levels are regulated by posttranslational mechanism in response to severe starvation stress and that the increased protein of GmbZIP53 under severe starvation accelerates transcriptional induction of GmASN1, GmProDH, and GmBCAT3. Furthermore, it is conceivable that decrease of branched chain amino acid level by the BCAT-mediated degradation eventually enhances autophagy under severe starvation.